RGS (regulators of G protein signaling) proteins are a family of modulatory proteins that sharpen G protein-coupled receptor signaling via their scaffolding and GTPase activating properties. Over 25 RGS subtypes are expressed in mammalian systems, and several are enriched in the nucleus accumbens, a critical neural substrate for drug reward. Since many drugs of abuse produce their effects via initial perturbation of G protein-coupled receptors, we and others have hypothesized that RGS proteins may play an important role in the brain's adaptive responses to drugs of abuse. We have obtained considerable evidence in support of this hypothesis in recent years. This work will now be further explored as part of this new Project. The RGS9-2 subtype is greatly enriched in nucleus accumbens and dorsal striatum. Given the high density of dopamine and opioid receptors in these structures, and the known modulation of Gi/o by RGS proteins, we have hypothesized that RGS9-2 is an important regulator of D2 dopamine and mu opioid receptors. Preliminary studies indicate that 1) RGS9-2 can modulate behavioral responses to cocaine and morphine, and 2) RGS9-2 expression in nucleus accumbens is regulated by chronic administration of these drugs of abuse. Certain other RGS subtypes that are expressed at high levels in this brain region, such as RGS4 and RGS2, are also regulated by drugs of abuse. We propose a series of highly integrated molecular, cellular, and behavioral studies to further elucidate the role of RGS9-2 and these other subtypes in the formation and maintenance of the addicted state. Specific Aim 1 will better define the influence of RGS9-2 on behavioral responses to cocaine and morphine. RGS9-2 activity will be manipulated in brain by use of viral vectors and transgenic mouse technology. A major goal of this Aim is to generate a floxed RGS9 mouse for region-specific and inducible deletion of the gene in brain. Aim 2 will clarify how cocaine and morphine regulate RGS9 levels. Analyses will determine the effects of dose, duration of administration, and mode of administration (forced vs voluntary) on RGS9-2 mRNA and protein levels. In addition, we will explore the effect of RGS9-2 phosphorylation on modulation of drug responses. Aim 3 will determine the cellular consequences of RGS9-2 activity using analyses of signal transduction pathways, cDNA micorarrays, and electrophysiological responses in nucleus accumbens neurons in brain slices and in Xenopus ooctyes. Aim 4 will begin to examine similar aspects of RGS2's and RGS4's involvement in addiction. The studies proposed in this Project will provide valuable clues to the role played by RGS proteins in drug abuse and addiction, and will inform the other Projects about the nature of adaptations in signaling pathways that drugs of abuse induce in the nucleus accumbens and other vulnerable neurons.